1a.Objectives (from AD-416)
(1) Investigate the molecular mechanisms that coordinate virulence and antibiotic resistance in Salmonella obtained from cattle (DT104 and S. dublin) and swine (DT104 and S. choleraesuis). (2) Characterize the molecular mechanisms involved in Salmonella survival, colonization and pathogenicity in relation to various host (swine and cattle) factors such as stress, gastric pH, and protozoa. (3) Investigate the molecular basis for swine resistance to Salmonella colonization by characterizing the immunological aspects of infection.

1b.Approach (from AD-416)
(1) Use a novel reporter system and transposon mutagenesis o identifiy DT104 genes involved in rumen protozoa-mediated hypervirulence. (2) Use an in vitro conjugation system to chracterize intra-protozoal transfer of plasmids from Klebsiella (and other bacteria) to Salmonella. (3) Compare the progression of salmonellosis for defaunated and fully-faunated calves infected with DT104. (4) Functional genomic analyses will be performed on Salmonella exposed to norepinephrine, low pH, and conditions relevant to intestinal colonization. (5) Use microarrays to identify swine genes that contribute to innate resistance to Salmonella colonization.

3.Progress Report
The level of stress hormones (e.g., norepinephrine) increases in pigs during stressful situations such as transportation and marketing. Since the gastrointestinal tract (the preferred invasion site for Salmonella) is highly innervated with the sympathetic nervous system, norepinephrine may serve as a potential signal to the pathogen of its in vivo environment. Therefore, investigation of the response mechanisms of Salmonella to norepinephrine may offer strategies for Salmonella intervention in the GI tract. Phenotypic microarrays performed on Salmonella exposed to norepinephrine have revealed potential systems in Salmonella to target for blocking the response of Salmonella to norepinephrine. Furthermore, in vivo investigation of the response regulator, QseB of the quorum sensing QseBC signal transduction system has been performed to analyze its role in swine pathogenesis. This work aligns with Component 1.2 of the National Program 108 Food Safety Action Plan and addresses Problem Statement 1.2.8 (Pathogenicity).

The susceptibility of an animal to Salmonella colonization and shedding can be a result of the host’s response to infection; thus, investigating the host’s response to Salmonella may identify genes that are responsible for the susceptibility, and thereby the Salmonella carrier-status, of the pig. To identify swine genes that have altered gene expression patterns between pigs that persistently shed Salmonella following experimental inoculation and those that do not, DNA microarray analysis was performed on whole blood of persistent shedders and non-persistent shedders of Salmonella, and real-time PCR is currently being completed to validate the microarray data. The goal of the project is to identify traits that could be exploited to enhance the resistance of swine herds to Salmonella colonization through immunomodulation, aid in the selection of breeder herds that have elevated resistance to Salmonella, and/or support the development of microbial diagnostic tools to identify Salmonella-carrier pigs. This work aligns with Component 1.1 of the National Program 108 Food Safety Action Plan and addresses Problem Statement 1.1.3 (Ecology, Host Pathogen and Chemical Contaminants Relationships).

4.Accomplishments
1.
A Mechanism for Porcine Immune Evasion by Salmonella Enterica Serovar Typhimurium.
Pigs that shed Salmonella in their feces are an animal health issue, a food safety problem and an environmental contamination risk; thus, identifying methodologies to control both clinical and sub-clinical (carrier) infections in swine is critical. To address this problem, the undesirable carrier state of Salmonella in pigs was investigated by examining the response of the gastrointestinal lymph nodes of pigs during infection with Salmonella enterica serovar Typhimurium. Our results indicate that a key immune response system (NFkappaB) is suppressed during infection. Suppression of the NFkappaB system may provide a strategy for Salmonella Typhimurium to evade a strong immune response by the pig. This immune evasion could contribute to the ability of Salmonella Typhimurium to establish a subclinical (carrier) infection in the pig. Thus, this research has identified a potential mechanism by which Salmonella Typhimurium eludes a strong inflammatory response to establish a carrier status in swine via suppression of the NFkappaB immune response system in antigen presenting cells. This work aligns with Component 1.1 of the National Program 108 Food Safety Action Plan and addresses Problem Statement 1.1.3 (Ecology, Host Pathogen and Chemical Contaminants Relationships).

5.Significant Activities that Support Special Target Populations
None.